Computational and experimental approaches to enhance extracellular secretion of recombinant proteins in Escherichia coli
Date
2010
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The annual global market for biopharmaceuticals is estimated at about $99 billion. Approximately 40% of all biotechnology products made in the US and EU are derived from Escherichia coli expression systems. Thus, efforts to study and improve E. coli as the host microorganism for the production of recombinant proteins can be of considerable importance. Secretion of recombinant proteins into the extracellular medium via the Type-I secretion system (TISS) in E. coli offers several advantages in comparison to the cytoplasmic accumulation and the periplasmic secretion such as simplifying detection and purification of the target protein and also providing a better folding environment. Studies have shown that the premature termination of the RutR protein, a transcription factor encoded by the ycdC gene, can enhance the secretion levels of recombinant proteins via the TISS. Here, the construction of an E. coli ΔycdC strain is described that enhances the extracellular levels of α-hemolysin (HlyA) and GILL-β- lactamase (GILL-Bla) (3.03-fold) proteins via the TISS. Previous experiments have shown that synonymous rare codon substitutions in the parent gene sequence can enhance the extracellular levels of multiple proteins via the TISS. Here, the combined effect of synonymous rare codon substitutions in the parent gene sequence and the ycdC gene knockout on the secretion levels of recombinant proteins via the TISS is investigated. The results suggest that the optimal levels of expression for an enhanced secretion of Bla and GILL-Bla proteins via the TISS may vary for the E. coli parent and the ΔycdC strain. In addition, synonymous rare rare codon substitutions in the predicted amphiphilic helix region of the C-terminal signal sequence can enhance extracellular levels of HlyA and Bla (2.07-fold) proteins via the TISS. These results describe the experimental strategies that can be used to enhance the secretion of recombinant proteins via the TISS. Next, two kinetic models of the TISS are illustrated that describe the secretion of the HasA protein from an E. coli parent and a ΔsecB strain. The SecB protein is a molecular chaperone required for efficient secretion of HasA via the TISS. The models incorporate multiple cellular events including HasA synthesis, protein folding, aggregation, chaperone interactions, degradation, transporter synthesis, reportertransporter interactions, catalytic cycle of the transporter, protein secretion into the extracellular medium and cell growth. The model simulation results are consistent with the qualitative trends in the experimental data available from the literature. A combination of techniques including parameter variation, sensitivity analysis, and nondimensionalization shows that the optimization of the extracellular concentration of HasA requires a balance between multiple factors including the HasA synthesis rate, the transporter synthesis rate and culture time. These results demonstrate the utility of a kinetic modeling approach towards enhancing extracellular recombinant protein secretion in E. coli.